Comparison of eight routine unpublished LC–MS/MS methods for the simultaneous measurement of testosterone and androstenedione in serum

• 8 routine unpublished LC-MS/MS methods for the simultaneous measurement of testosterone and androstenedione were compared.
• Some of the investigated methods in the present study showed a good agreement.
• There are differences in standardization between some of the assays and some assays show a high variation or low recoveries.
• The overall agreement of unpublished routine methods compare unfavourably to the variation found in published methods.
• These findings stress the importance of thorough initial validation but also for the continuous monitoring of LC-MS/MS methods.
• The policy to require the use of LC-MS/MS assays alone is not an assurance for high quality sex steroid measurement results.

BackgroundLiquid-chromatography tandem mass spectrometry (LC–MS/MS) has become the method of choice in steroid hormone measurement. However, little information on the mutual agreement of LC–MS/MS methods is available. We compared eight routine unpublished LC–MS/MS methods for the simultaneous measurement of testosterone and androstenedione.MethodsSixty random serum samples from male and female volunteers were analysed in duplicate by eight routine LC–MS/MS methods. We performed Passing–Bablok regression analyses and calculated Pearson's correlation coefficients to assess the agreement of the methods investigated with one published method known to be accurate. Intra-assay CV of each method was calculated from duplicate results, recoveries for each method were calculated from six spiked samples. Furthermore, a CV between the investigated methods was calculated.ResultsThe concentrations ranged from 0.05–1.26 nmol/L, 6.15–24.44 nmol/L and 0.15–4.78 nmol/L for testosterone in females, testosterone in males and androstenedione, respectively. The intra-assay CVs were between 3.7–16.0%, 0.9–5.2% and 1.2–9.5% for testosterone in females, testosterone in males and androstenedione, respectively. The slopes of the regression lines ranged between 0.90–1.25, 0.87–1.24 and 0.94–1.31 for testosterone concentrations in females, all testosterone values and androstenedione, respectively. Inter-method CVs were 24%, 14% and 29% for testosterone for concentrations in females and males and androstenedione, respectively. These compare unfavourably to the variation found earlier in published methods.ConclusionAlthough most routine LC–MS/MS methods investigated here showed a reasonable agreement, some of the assays showed a high variation. The observed differences in standardization should be taken into account when applying reference values, or should, preferably, be solved.